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Introduction:
Also
known as Barbados Cherry, West Indian Cherry, Cereza, Cerisier and
Semeruco. Phenolic compounds are important for plant metabolism and
have also becoming important for humans due to their health
characteristics, particularly related to their antioxidant
properties.Anthocyanins are one of the most attractive plant
phenolic pigments of the group of flavonoids. Their visual impact
allied to their health properties make them potentially useful as
natural food colorants. One possible source of anthocyanin pigments
is the
fruit
of acerola (Malpighia punicifolia, L.), a highly productive plant,
which provides fruits during long periods
of the year and is extensively cultivated in Brazil.
The attractive red colour in acerola skin is mainly due to
anthocyanins and the abundance of this fruit in Brazil represents a
potential source of anthocyanin pigments.
Flavonoids are polyphenolic compounds that occur ubiquitously in
food of plant origin and more than 2000
chemically distinct flavonoids have been already reported.
Phenolic compounds are also important because of their contribution
to the sensory quality of fruits (colour and flavour, including
astringency and bitterness) which may be affected during the
technological processes used for production of juice and other
derived products.3 In
addition, anthocyanins and other phenolic compounds have been used
successfully in the characterization of fruits and juices.
Due
to the importance of flavonoids, many techniques have been used to
identify and quantify these compounds.In the early 1990s,
high-performance liquid chromatography (HPLC), with photodiode array
detection was used to isolation and quantification.1H and 13C NMR
spectroscopy are the most powerful techniques for determination of
molecular structure. The coupling of HPLC and mass spectrometry
methods, such as electrospray, thermospray, or fast-atom
bombardment, have been widely used to provide molecular weight and
characteristic fragment ions for structural elucidation2, but these
are very expensive techniques which are not readily available.5 Due
to simplicity of methodology and low cost, techniques like paper
chromatography and thin-layer chormatography have been applied
routinely in many laboratories.2,6-8 Acerola skin is a byproduct of
acerola processing usually discarded in juice and pulp industries
and due to its relatively high levels of anthocyanins it could be
exploited as a potential source of these pigments. The interest in
identifying sources of anthocyanin pigments to 665 Phenolic
Compounds in Acerola Fruit (Malpighia punicifolia, L.) be used in
food, pharmaceutical and cosmetic industries is based on consumer
demand for natural colorants, and on the nutraceutical properties
frequently reported for flavonoids.9 However, as far as we know, no
studies on characterization of acerola phenolics have been
published. Thus, the objective of the present work was to isolate
and characterize the phenolic compounds present in acerola and to
estimate the amount of anthocyanin pigments found in the acerola
skin which is usually a by product from the production of acerola
pulp.
Material and Methods:
Fruit
Acerola fruits (M. punicifolia, L.) were chosen by intensive red
colour, firmness and a pleasant acid flavour, which are indicative
of an ideal stage of ripeness. The fruits were harvested during June
2000 in a commercial
plantation in Rio de Janeiro city (Brazil). After harversting, the
fruits were washed, packed in polyethylene bags and stored in the
dark at –18 °C for two days before anthocyanin extraction. Chemicals
Caffeic, ferulic, p-coumaric and chlorogenic acids and quercetin,
kaempferol, myricetin, and pelargonidin chloride, were obtained from
Sigma Chemical Co.
The
solvents used were of HPLC grade obtained from Tedia Co. All other
chemicals were of analytical grade. HPLC equipment A Pharmacia
(Switzerland) liquid chromatographic system, equipped with two pumps
(LKB model 2248), a controller (LKB model LCC 2252), and an injector
motor valve (model PMV-7) with 20 µL loop was used. The system was
attached to a UV-VIS detector SPD 10 AV. An integrator model C-R6A
was used for data processing.Characterization of anthocyanin
aglycones
Anthocyanins were analyzed in the extract from acerola skin where
these pigments are concentrated. The skin of ripe acerola fruit
(500g) was extracted with 600 mL of acidified methanol (0.1% citric
acid, v/v) in the dark at about 15 °C and the extract concentrated
under vacuum.
Due
to the non-availability of standards, anthocyanin characterization
was carried out based on several physicochemical information such as
mobility in HPLC, separation by paper chromatography and
spectroscopic characteristics. Paper chromatographic separation was
performed in the dark using Whatman paper 3 MM Chr (23 x 57 cm)
according to the method described by Francis.10 Analysis of HPLC was
performed in a Lichrospher-100 RP-18 column (250 x 4.6 mm) using
methanolwater
(1:1, v/v) acidified with 2 mol L-1 HCl as mobile phase until pH 2.5
and flow rate of 1.3 mL min-1. Detection was carried out at 530
nm.11 UV-Vis spectroscopy was carried out in the fractions separated
by paper chromatography that were dissolved in methanolic 0.01% HCl.
UVVis
spectra were obtained with a Beckman DU 650 spectrophotometer
,
in the range of 200-600 nm, with the absorption spectra registered
before and after the addition of 3 drops of a solution of the
aluminum chloride
salt in absolute ethanol (5% m/v).8,12-14 The position of the sugar
in the anthocyanin molecule was assigned based on the spectral
shifts by calculation of the ratio of absorption at 440 nm to the
specific absorption maximum for each pigment.8,13,14 The amount of
total anthocyanin in the
acerola skin was estimated based on the calculation described by
Lees and Francis15 after measurement of the absorbance of the
acidified ethanol extract at 535 nm. Characterization of flavonols
and phenolic acids
Flavonols and phenolic acids are distributed both in the fruit skin
and pulp.
Consequently, in order to obtain a higher yield, the whole fruit was
used for extraction. Frozen acerola fruit (260 g) was blended with
250 mL of cooled
distilled water and centrifuged at 3000 x g for 20 min at 4 °C. The
extraction and centrifugation procedures were repeated twice. The
combined supernatants were filtered through Whatman No.1 filter
paper. Selective solvent extractions were then carried out to obtain
one fraction containing the flavonol aglycons and other containing
the phenolic acids, following the procedure described by Kader
et
al.16
Characterization was then carried out using HPLC for separation and
identification was done by comparison and spiking with external
standards. For the analysis of flavonol aglycons, the mobile phase
was methanol containing 0.5% orthophosphoric acid (1:1 v/v) with a
flow rate of 1.2 mL/min., and detection at 370 nm.17 For the
analysis of the phenolic acids, the mobile phase consisted of a
gradient elution with 0.01 mol L-1 tri-sodium citrate solution and
20% methanol adjusted to pH 2.5 with 6 mol L-1 HCl (solvent A) and
methanol (solvent B). Elution was performed at a flow rate of 1.0 mL/min
with the gradient of 5 min for B, increasing to 19% in 15 min, 20%
in 5 min, 30% in 10 min and 50% in 10 min.at 325 nm.18 In both
cases, a Lichrospher-100 RP-18 column was used.
Results and Discussion:
The
main objective of the present study was to characterize the phenolic
compounds present in acerola, including the aglycones of phenolic
glycosides. The structures of the compounds studied are shown in
Figure1. Phenolic pigments such as anthocyanins are usually found as
glycosides in plants, however, when they are ingested as food the
sugars are easily hydrolyzed from the aglycones. HPLC analysis of
the anthocyanin fraction showed three peaks with retention times of
5.7 min, 14.9min and 22.1 min (Figure 2). Similarly, paper
chromatography revealed three bands with pink, magenta and orange
colours, respectively. The typical order of
chromatographic elution in reverse phase HPLC of different
anthocyanins with similar glycosylation patterns is determined by
the polarity of the aglycone. The three peaks observed in the HPLC
chromatogram were correspondent to the three bands observed by
descending paper chromatography. Peak 1 corresponded to the pink
band (Rf = 0.43); peak 2 corresponded to the magenta band (Rf =
0.12) and peak 3 to the orange band (Rf = 0.56). The different
colours of anthocyanin pigments reflect the nature of their
hydroxylation and methoxylation patterns. An increase in
hydroxylation is accompanied by an increase in blue colour while
methoxylation enhances the red colour.1 This would indicate that
peak 1 may correspond to a malvidin with one methoxylation and peak
2 to a cianidin with two hydroxylations. Peak 3 indicates one
hydroxylation in the molecule which confirms the assignment of
pelargonidin that was determined by comparison and spiking with the
external standard. Additional qualitative information was obtained
with the aid of the spectral characteristics of the chromatographic
bands. The spectral data presented in Table 1 show the maximum
absorptions for the chromatographic peaks
as
277 and 536 nm for peak 1 (5.7 min); 281 and 527 nm Figure 1.
Chemical structure of phenolic compounds identified in acerola.
their antioxidant properties.9,22-24 The fraction containing
flavonol aglycons showed as expected maximum absorbance at 370 nm.
Individual peaks obtained by HPLC were assigned after comparison of
retention times and coelution with flavonol standards showing the
presence of quercetin (Rt 8.2 min) and kaempferol (Rt 13.0 min)
In
conclusion, the phenolic compounds detected in acerola may be
classified in two categories; phenolic anthocyanin pigments and non-anthocyanin
phenolics. The pigments detected were a 3,5-diglycosilated malvidin,
a 3-monoglycosilated cyanidin and pelargonidin. Nonanthocyanin
phenolic compounds identified were pcoumaric acid, ferulic acid,
caffeic acid, chlorogenic acid,
kaempferol and quercetin.
However, in the presence of acerola cherry extract, both soy and
alfalfa extracts potently inhibited the formation of LDL-. These
findings show that acerola cherry extract can enhance the
antioxidant activity of soy and alfalfa extracts in a variety of LDL
oxidation systems. The protective effect of these extracts is
attributed to the presence of flavonoids in soy and alfalfa extracts
and ascorbic acid in acerola cherry extract, which may act
synergistically as antioxidants. It is postulated that this
synergistic interaction among phytoestrogens, flavonoids, and
ascorbic acid is due to the "peroxidolitic" action of ascorbic acid,
which facilitates the copper-dependent decomposition of LDL
peroxides to nonradical products; this synergy is complemented by a
mechanism in which phytoestrogens stabilize the LDL structure and
suppress the propagation of radical chain reactions. The combination
of these extracts markedly lowers the concentrations of
phytoestrogens required to achieve significant antioxidant activity
toward LDL.
Acerola, also known as Barbados cherry or West Indian cherry, is
grown to a minor extent in the frost-free regions of Florida and in
Hawaii, primarily in home gardens (Miller et al. 1965). This plant
is most noted for the extremely high ascorbic acid (vitamin C)
content of its fruit, with 10 to 40 mg/g of edible fruit, far more
than any other known fruit. By comparison, The juice retains its
cherry-red color and flavor if it is processed and frozen
immediately. The development of a chemical method of producing
vitamin C has reduced the need for acerola.
The
acerola is believed to originate from the Yucatan and can be found
growing in the sandy soils of Mexico, Central America, northern
South America (Venezuela, Surinam, Columbia) and throughout the
Caribbean (Bahamas to Trinidad). Acerola has now been successfully
introduced in sub-tropical areas throughout the world (Southeast
Asia, India, South America), and some of the largest plantings are
in Brazil.
The acerola is a deciduous tree typically found in dry woodlands. It
has poor cold tolerance, with young plants typically killed at
temperatures below 30°F - trees can survive brief exposure to 28°F,
but will lose leaves. They are also sensitive to wind as they have
shallow root systems, but are drought tolerant, and will adopt a
deciduous habit.
The acerola tree is a large, relatively fast growing bushy shrub or
small tree it can grow up to 15 feet). The branches are brittle, and
easily broken. Acerola leaves are dark to light green and glossy
when mature. They are obviate to lanceolate, with minute hairs which
can be irritating to some people. The flowers are small, pink to
white in colour and have five petals. Acerola fruit are round to
oblate and cherry-like, but with 3 lobes. They are bright red
(rarely yellow-orange) with thin skin, and are easily bruised. The
pulp is juicy, and quite acidic with a delicate flavour, and apple
notes.
The fruit of the Acerola Cherry tree, Malpighia punicifolia L. is
rich in Vitamin C and carotenoids, with the cherry-like fruits being
one of the richest known natural sources of vitamin C. The fresh
fruit can contain up to 4000mg Vitamin C per gram of fresh weight
(although typically, it is around 1500mg). Oranges provide 500 to
4,000 parts per million Vitamin C or ascorbic acid, while Acerola
assays in the range of 16,000 to 172,000 parts per million. Green
fruits have twice the Vitamin C level of mature fruits. Fruits
develop to maturity in less than 25 days.
Acerola also contains the synergistic bioflavonoids - rutin and
hesperidin, carotenoids, and other vitamins, minerals and
phytonutrients, making it an ideal food based source of nutrients
necessary for immune support. Compared to oranges, acerola provides
twice as much magnesium, pantothenic acid, and potassium. Other
vitamins present include vitamin A (4,300 to 12,500 IU/100g),
thiamine, riboflavin, and niacin in concentrations comparable to
those in other fruits. One hundred and fifty other constituents have
been identified in acerola; the major ones being furfural,
hexadecanoic acid, and limonene. Aside from being an excellent
source of powerful antioxidants, Acerola cherries are also rich in
protein and mineral salts - principally iron, calcium and
phosphorus.
Recent research in cosmetology indicates that vitamin C is a
powerful antioxidant and free radical scavenger for the skin, and
acerola extracts are now appearing in skin care products that fight
cellular aging. The mineral salts contained in acerola have also
been shown to aid in the re-mineralisation of tired and stressed
skin, while the mucilage and proteins have skin hydrating
properties, and promote capillary conditioning.
Acerola cherry :
Acerola has been studied in the laboratory and has been found to be
a powerful antioxidant and have anti-tumor potential.
Acerola cherry flavor:
Volatile components have been isolated from acerola fruit. One
hundred fifty constituents have been identified in the aroma
concentrate, from which furfural, hexadecanoic acid,
3-methyl-3-butenol, and limonene were found to be the major
constituents. The amounts of esters, 3-methyl-3-butenol, and their
various esters are thought to contribute to the unique flavor of the
acerola fruit.
Acerola cherry Research Update
Antioxidant activity of dietary fruits, vegetables, and commercial
frozen fruit pulps. Fruits, vegetables, and commercial frozen pulps
(FP):
consumed
in the Brazilian diet were analyzed for antioxidant activities using
two different methods, one that determines the inhibition of
copper-induced peroxidation of liposome and another based on the
inhibition of the co-oxidation of linoleic acid and beta-carotene.
The anthocyanin-rich samples showed the highest,
concentration-dependent, antioxidant activities in both systems. In
the liposome system, at both 10 and 50 microM gallic acid equivalent
(GAE) addition levels, the neutral and acidic flavonoids of red
cabbage, red lettuce, black bean, mulberry, Gala apple peel,
jambolao, acai FP, mulberry FP, and the acidic flavonoids of acerola
FP showed the highest antioxidant activities (>85% inhibition). In
the beta-carotene bleaching system, the samples cited above plus red
guava gave inhibition values >70%. On the other hand, some samples
showed pro-oxidant activity in the liposome system coincident with a
low antioxidant activity in the beta-carotene system. There was no
relationship between total phenolics content, vitamin C, and
antioxidant activity, suggesting that the antioxidant activity is a
result of a combination of different compounds having synergic and
antagonistic effects.
Structural and functional characterization of polyphenols isolated
from acerola (Malpighia emarginata DC.) fruit:
Two
anthocyanins, cyanidin-3-alpha-O-rhamnoside (C3R) and
pelargonidin-3-alpha-O-rhamnoside (P3R), and quercitrin
(quercetin-3-alpha-O-rhamnoside), were isolated from acerola (Malpighia
emarginata DC.) fruit. These polyphenols were evaluated based on the
functional properties associated with diabetes mellitus or its
complications, that is, on the radical scavenging activity and the
inhibitory effect on both alpha-glucosidase and advanced glycation
end product (AGE) formation. C3R and quercitrin revealed strong
radical scavenging activity. While the inhibitory profiles of
isolated polyphenols except quercitrin towards alpha-glucosidase
activity were low, all polyphenols strongly inhibited AGE formation.
[Physico-chemical characterization of acerola (Malpighia glabra L)]:
The
acerola Malpighia glabra L., originally from the Antillas and North
of South America, known by the people as cereja-das-antilhas or
cereja-do-para distinguish itself by its high content of vitamin C.
The ripe and fresh acerola fruits utilized in experiments, were
obtained from farmers of Maringa region, Parana State, Brazil. The
fruits were hulled in steel sieve with 25 mesh and the bagasse
(seeds and hull) discarded. These physico-chemical analysis were
realized in the pulp: vitamin C, moisture, protein, carbohydrate,
fiber, lipids and fatty acids composition. We also determined the
content of ash and cadmium, calcium, lead, copper, chrome, iron,
magnesium, manganese, potassium, sodium and zinc minerals. The
average content of vitamin C was 1.79 g/100 g of pulp, it was higher
than the one for other fruits, like pineapple, araca, cashew, guava,
kiwi, orange, lemon, and strawberry and lower than the camu-camu
sylvestral fruit of Amazonia. The contents of moisture,
carbohydrate, fiber, lipidsandminerals in the acerola were not
significantly different when compared to other fruits. |
